Human immunodeficiency virus type 1 (HIV-1) has four genes, vif, vpr, vpu, and nef; termed “accessory genes,” that are dispensable for viral replication ill vitro (14). Many important functions related to HIV-1 pathogenesis have been ascribed to these accessory genes. Specifically, Vpr has been implicated in long terminal repeat transactivation, nuclear import of the preintegration complex, induction of G2 cell cycle arrest, and apoptosis. Recent studies identifying single amino acid changes in Vpr in a cohort of HIV-1-infected long-term nonprogressors substantiated the role of Vpr in HIV-1 pathogenesis in vivo (29, 43). Vpr induces G2 arrest and apoptosis in infected CD4+ lymphocytes (18, 21, 35, 38). G2 arrest by Vpr is effected in HeLa cells through activation of the ATR-dependent DNA damage checkpoint pathway (40). This data supports previous work demonstrating the inhibition of cyclin B1-p34cdc2 complexes by Vpr (18) and establishes the identity of some of the upstream regulators of Cdc2. ATR-dependent activation of Chk1 kinase leads to the inhibition of Cdc25C phosphatase, which is normally required to dephosphorylate and activate Cdc2 (40). The signaling pathway downstream of ATR activation was recently reviewed in references 1 and 33.
While Vpr activates the ATR-specific checkpoint, the role of other molecules required in the ATR pathway is not known. Activated ATR can also phosphorylate proteins other than those required for G2 arrest. One of these substrates is the histone 2A variant X (H2AX). H2AX is deposited randomly throughout chromatin, comprising approximately 10% of total nucleosomal histone H2A (34). H2AX has a highly conserved serine residue at position 139 that is phosphorylated by ATR and/or ATM in response to DNA damage (10, 37, 46). It is estimated that hundreds to thousands of H2AX molecules are phosphorylated per double-stranded break (37). ATM-dependent H2AX phosphorylation occurs in response to doublestranded DNA breaks (10, 46, 47). In contrast, ATR phosphorylates H2AX under circumstances of replication stress, such as stalled replication forks (9). In the presence of DNA damage or replication stress, H2AX molecules that are located in the vicinity of the DNA lesion become phosphorylated in a highly specific localized manner (34). Thus, immunofluorescence staining for phosphorylated H2AX (also referred to as γ-H2AX) following DNA damage produces a staining pattern of distinct nuclear foci (34). γ-H2AX is thought to amplify the DNA damage signal by enhancing and stabilizing the recruitment of DNA damage sensor proteins, such as ATR, ATM, Rad17, and the 9-1-1 complex, and DNA repair proteins, such as breast cancer susceptibility protein 1 (BRCA1), Nbs1, Mre11, and Rad50, to sites of DNA damage (15). This action may effectively “mark” the site of DNA damage, maintaining checkpoint signaling at the damaged region until DNA repair is completed.
Another substrate of activated ATR is BRCA1. BRCA1 is important for both checkpoint activation and DNA repair. BRCA1 colocalizes with DNA repair factors, such as Rad51, PCNA, and Mre11-Rad50-Nbs1 (15). It has been proposed that BRCA1 may represent an essential link in coordinating cell cycle arrest with genomic repair efforts (reviewed in reference 27) and with the induction of apoptosis.
In addition to a role in cell cycle arrest, Vpr plays a role in apoptosis. However, it is not possible to extrapolate the findings relating to cell cycle arrest to apoptosis, as the pathways do not completely overlap or follow one from the other. Therefore, there is also a need to determine the role of Vpr in apoptosis.
It has been suggested that apoptosis of infected cells may play a significant role in the depletion of CD4+ lymphocytes in vivo (62, 82, 56, 93). However, the mechanism by which Vpr induces apoptosis was not understood. Muthumani et al. reported that vpr-expressing cells undergo apoptosis via the intrinsic pathway that involves loss of mitochondrial membrane potential (74). This pathway of apoptosis is characterized by cytochrome C release, and caspase 9 activation, and is triggered in the absence of death receptor ligation (74). However, the initial event induced by Vpr towards activation of the proapoptotic signaling cascade was not elucidated.
To elucidate whether Vpr might directly promote the release of pro-apoptotic mediators from the mitochondria, Veira et al., and Jacotot et al. incubated recombinant Vpr with purified mitochondria (88, 67). These two studies found that in a cell-free system, Vpr interacts with the permeability transition pore complex (PTPC) to cause ion permeability and swelling of mitochondria leading to release of cytochrome C (88, 67). These results support a model in which Vpr induces mitochondrial depolarization directly rather than activating upstream stress signals (88, 67). The present invention provides data that does not support the model of Jacotot et al. and provides additional methods of activating apoptosis.